Introduction: actin and myosin Actin Myosin Myosin V and actin 375 residues Found in all eukaryotes Polymeric Forms track for myosin Many other cellular functions 36 nm pseudo-helical repeat Catalytic domain: ~750 res. Entire protein: ~1800 res. Binds 2-6 light chains (calmodulin) Found in almost all eukaryotes Hydrolyzes ATP to generate motion Myosin V: 6 light chains, coiled-coil domain, and globular tail domains Lever arm swing generates motion Other myosins function as monomers, dimers, or filaments Alex Dunn, Sept. 2005
Myosin evolution Hodge et al. J. Cell Sci. 2000, 113, 3353. Richards et al. Nature, 2005, 436, 1113.
Characterized myosins Myosin II Conventional myosin by default. The myosin in skeletal muscle. Also responsible for pinching off cell during cytokinesis. Commonly studied myosin II varieties polymerize into filaments with thousands of heads. Binds 2 light chains. Cardiomyopathy mutations, also congestive heart failure, cancer. Myosin V Moves cargo around cell: vesicles, RNA. Binds 6 light chains, and forms a tight dimer. Walks hand-over-hand, with many consecutive steps before detaching. 36 nm step: 1 actin pseudo-repeat. Rare hereditary disease. Myosin VI Moves cargo around cell. Important in inner ear hair cell formation. Walks in the opposite direction of other myosins, toward (-) or pointed end of the actin filament. Probably functions as a dimer, but dimerizes weakly by itself. Binds only 2 calmodulins, but dimeric constructs take 36 nm steps. Rare hereditary deafness
Lever arm hypothesis Proposed by Hugh Huxley, 1969 Supported by single molecule spectroscopy, x-ray crystallography, FRET, electron microscopy Consensus model. Dissenting model proposed by Yanagida: Tanaka, Nature. 2002, 415, 192. Spudich, J.A. Nature Rev. Mol. Cell Biol. 2001, 2, 387. Purcell, et al. Proc. Natl. Acad. Sci. USA 2002, 99, 14159.
Example experimental data FRET Optical Trapping Shih, et al. Cell, 2000, 102, 683. Mehta et al. Nature, 1999, 400, 590.
Example experimental data Single molecule Fluorescence Random or directed mutations Below: result of a screen for Dicty mutants with temperature-sensitive myosin function. Yildiz et al. Science 2003, 300, 2061. Ökten et al. Nature Struct. Mol. Biol. 2004, 11, 884. Patterson, et al. Genetics 1996, 143, 801. Sasaki et al. Biochemistry 2003, 42, 90.
Example experimental data Bulk kinetics Other techniques Cryo-electron microscopy Muscle fiber pulling Fiber x-ray diffraction EPR spin labels Mass spectrometry Crystallography (next slides) De La Cruz, et al. Proc Natl. Acad. Sci. USA 1999, 96, 13726. and many, many others
Known myosin conformations 1) Pre-stroke Smooth muscle ADP BeF x, AlF x : 1BR2 Dicty ADP VO 4 : 1VOM Scallop ADP VO 4 : 1QVI - Lever arm cocked, pre-stroke - Cleft Open 2) Rigor-like Myosin V no nucleotide, ADP: - Lever arm post-stroke - Closed cleft consistent with cryo-em structures of Actin-myosin complex. 1OE9 Dicty-GTPase fusion*, no nuc.: 2AKA - Not as closed as 1OE9 3) Post-Rigor Scallop no nucleotide: Dicty ADP: Myosin V ADP BeF x : - Lever arm post-stroke 1KK7 1MMA 1W7J - Cleft open: after release from actin, but before ATP hydrolysis Post-post-stroke state (?) Scallop ADP extended past rigor: 1B7T - Lever arm swung past post-stroke angle. - Cleft open - Significance uncertain. * Dicty myosin fused to a GTPase to get the GTPase to crystallize. Novel myosin structure was fortuitous, and is only observed with the fusion. However, it is structurally similar to 1OE9.
Large Domains Myosin parts list Smaller Elements N-terminal Contacts coverter in post-stroke state (?) Upper 50 kd domain Binds, hydrolyzes ATP Lower 50 kd domain Converter Lever Arm Binds actin Fulcrum, connects to lever arm Amplifies motion, binds light chains Switch I loop Communicates actin binding state to nucleotide pocket (?) Switch II loop P-loop Communicates nucleotide state to lever arm ATP binding and hydrolysis (?) Relay Helix Undergoes large conformational shifts between pre, post-stroke states SH1 and SH2 helices Communicate nucleotide state to lever arm
Switch I and Switch II Switch I Closed = bound nucleotide, Mg 2+ Open = no nucleotide Alanine scanning shows switch I is essential for in vitro function Shimada Biochemistry 1997, 36, 14037. Structures of apo Dicty and chicken myosin V cited as showing switch I in an open conformation. Reubold, Nature Struct. Biol. 2003, 10 827. Coureux EMBO J. 2004, 23, 4527. In the manuscript Coureux et al. say that active site changes due to P loop, not switch I. Communication between switch I and the upper 50 kd (actin binding) domain (?) Switch II Closed = pre-stroke and rigor Open = post-rigor Mutagenesis shows switch II is essential for both ATP hydrolysis and communication with the lever arm. Sasaki, J. Biol. Chem.1998, 273, 20334. Murphy Nature Cell Biol. 2001, 3, 331. Best example: scallop structures show switch II in open, closed conformations. Gourinath Structure 2003, 11, 1621. Houdusse Cell 1999, 97, 459. Bagshaw group tryptophan fluorescence papers (Switch I, II) Zeng Phil. Trans. R. Soc. B 2004 359, 1843. Malanasi-Csizmadia J. Mus. Res. Cell Motil. 2005 online ed.
One detailed (among several) myosin mechanism A = Actin M = Myosin o = open (switch I, switch II) T = ATP D = ADP P = phosphate Zeng Phil. Trans. R. Soc. B 2004 359, 1843.
The order of the power stroke, Pi 1) Multiple myosin ADP/actin states can be kinetically distinguished release, and Mg 2+ release - Rosenfeld, J. Biol. Chem. 2000, 275, 25418. 2) Pi diminishes force generation - (Millar & Homsher 1990; Dantzig et al. 1992; Walker et al. 1992) 3) The initial interaction between myosin and actin is weak and salt sensitive 4) ADP release is rate limiting - Siemankowski et al. 1985 5) Recent results: Mg 2+ release weakens the myosin ADP complex. -Rosenfeld J Biol. Chem. 2005, 280, 6072. Mg 2+ release (?) power stoke may begin with Pi release Refs. in parentheses from: Takagi et al. Phil. Trans. R. Soc. B 2004 359, 1913.
Myosin thermodynamics 1) ATP binding is a large source of potential energy: K d ~ 10-10 M, ~60 kj/mol. Goody et al. Eur. J. Biochem. 1977, 78, 317. 2) The post-stroke myosin binds actin quite tightly: ~ 30 kj/mol. 3) ATP hydrolysis is reversible: ~ 0 kj/mol Trade ATP for actin binding G ~ 0 Weak to strong binding coupled to Pi release, power stroke Bagshaw et al. Biochem J. 1973, 133, 323. 4) Maximum work done by myosin: 30-40 kj/mol. Karatzaferi, et al. Biophysical J. 2004, 87, 2532. Cooke, J. Gen. Physiol. 2004, 123, 643.
Supplemental: myosin screen shots blue: N-terminal domain purple: upper 50 kd domain red: lower 50 kd domain green: converter red: nucleotide green: P-loop orange: switch I yellow: switch II cyan: SH1 helix light blue: SH2 helix purple: relay helix/loop Note: Pictures are rotated 180 degrees about the vertical with respect to each other